Snapin Regulates The Membrane Transport Process By Blocking The Endoplasmic Reticulum Of Cav1.3 Calcium Channel

It has recently been established that Cavl.3(α1D) variant L-type Ca2+channels palys particular role in generating electrical activity in atrial and cardiac pacemaker cells. However, the molecular and functional basis of Cavl.3Ca2+channel modulation is not yet fully understood. Here we reported that a protein associated with C terminal of Cav1.3Ca2+channel was screened from human cardiac cDNA library by using yeast two hybrid system. The positive protein was identified as snapin after sequencing. The physical interaction between snapin and Cavl.3Ca2+channel proteins was confirmed by co-immunoprecipitation assays in HEK293cells co-expressing snapin and Cav1.3Ca+channels and in cardiac myocytes isolated from mouse, rat as well as human atrium. In addition, co-localization of two proteins at cells surface in either cultured HEK293cells or cardiac cells isolated from mouse atria was addressed by immunocytochemical detection. GST pull down assay showed that snapin through its83～136aa region (a coil-coil domain) directly binds to the C terminal of Cav1.3Ca2+channel in vitro. Strikingly, the functional study demonstrated that whole cell current densities recorded from the HEK293cells co-expressing snapin and Cavl.3Ca2+channels were significantly smaller than those cells expressing Cavl.3alone. In consistance with patch claming data, a marked reduction in both total and membrane expression of Cavl.3protein in response to over-expression of snapin was evidenced by western blotting analysis. Furthermore, over-exression of snapin did not affect Cavl.3expression at mRNA level as revealed by RT-PCR detection, rather, snapin-associated ER retention of Cavl.3, the enhanced ubiqutination of Cavl.3, and the degradation of Cav1.3through proteasome were documented by immunofluoresnece staining and western blotting analysis. Interestingly, it was found that snapin is able to form a ternary complex with SNAP-23and Cavl.3. The site of Cavl.3snapin binds to is the same site where SNAP-23usually associated with to facililitate channel expression on the cell membrane. With the increase of snapin expression, binding of SNAP-23to Cavl.3was notablely diminished, suggesting a compitition between snapin and SNAP-23. Taken together, our data, for the first time, prove that snapin competitively interferes with the binding of SNAP-23to the C terminal of Cavl.3Ca2+channel and then causes an obstruction of Cavl.3exporting from ER. As a result, ER-retained Cavl.3proteins were degradated by ubiquitin proteasome pathway, leading to a significant reduction in total and menmbrane expression of Cav1.3protein. However, it is necessary to further investigate how snapin works in coordination with SNAP-23. to modulate the expression and function of Cavl.3Ca2+channels.